The HIV-1 epidemic has resulted in ~2.7 million new infections in 2007 for a total of ~33 million people living with HIV/AIDS. Clinical trials have shown that HIV-1 infection cannot be prevented by immunization with monomeric recombinant forms of viral envelope (Env) proteins. However, it is clear that the HIV-1 Env contains epitopes that can induce neutralizing antibodies and that such antibodies can protect primates from infection. There are several reasons why monomeric gp120 has been ineffective at inducing neutralizing antibodies in previous animal and human studies. The surface-exposed variable sequences surround the CD4 binding site and might restrict B-cell access to this epitope. The gp120 protein carries epitopes such as the V3 loop that are immunogenic but not exposed to antibodies in the native virus Env structure. Two main hypotheses inform the design of immunogens for HIV-1 vaccines. The first hypothesis focuses on broadly neutralizing epitopes defined by monoclonal antibodies. It is hypothesized that suitable immunogens will expose these epitopes and induce polyclonal antibodies with neutralizing specificities comparable to those of the monoclonals. The second hypothesis suggests that a single broadly cross-reactive neutralizing specificity is not be readily induced and that multiple epitopes must be recognized to inhibit virus infectivity. Both hypotheses can be tested by the design and creation of Env-based immunogens lacking variable sequences that might be immunodominant and in any case cannot induce broad neutralizing activity. Removal of variable regions while retaining CD4 binding activity might expose this important epitope as well as others that reside in the relative conserved regions of Env. This approach represents an initial step in a recursive process to build upon a concept, or design, of what features an immunogen should possess. However, experimental testing of a single prototype sequence does not allow a robust evaluation of the concept. An alternative approach, used in this Proposal, is to create sequence variants for a given vaccine concept and to test a number of those variants. In this way, a larger and more significant dataset can be obtained from which to draw conclusions regarding the value of the immunogen design. In this Proposal, we will attempt to overcome some of the limitations of gp120 as an immunogen. We recognize that an immunogen format such as a soluble trimer mimic or a virus-like particle carrying gp120 might ultimately be required for an effective Env-based vaccine. The specific aims of this Proposal are as follows: (1) incorporate modifications into gp120 and evaluate their effect on ligand binding properties;(2) create combinatorial libraries of gp120 variants and identify those with CD4 binding activity;(3) immunize rabbits with selected gp120 variants and evaluate the neutralizing activity If the gp120 variants are able to induce neutralizing antibodies, then additional studies will form the basis of a Phase II SBIR proposal. PUBLIC HEALTH RELEVANCE: The HIV/AIDS epidemic has resulted in 2 million deaths and 2.7 million new infections in 2007, for a total of nearly 33 million people living with HIV/AIDS. Development of a vaccine is considered to be an essential component of the public health measures needed to slow the epidemic. This research proposal is designed to create a vaccine that can induce antibodies capable of preventing infection by the HIV virus.